International Journal of
Molecular Sciences
Article
Exploratory Characterization of Phenolic Compounds
with Demonstrated Anti-Diabetic Activity in Guava
Leaves at Different Oxidation States
Elixabet Díaz-de-Cerio 1,2 , Vito Verardo 3, *, Ana María Gómez-Caravaca 1,2 ,
Alberto Fernández-Gutiérrez 1,2 and Antonio Segura-Carretero 1,2, *
1
2
3
*
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd. Fuentenueva s/n,
18071 Granada, Spain; [email protected] (E.D.-d.-C.); [email protected] (A.M.G.-C.);
[email protected] (A.F.-G.)
Functional Food Research and Development Center, Health Science Technological Park,
Avd. del Conocimiento, Bioregion building, 18100 Granada, Spain
Department of Chemistry and Physics (Analytical Chemistry Area) and Research Centre for Agricultural
and Food Biotechnology (BITAL), Agrifood Campus of International Excellence, ceiA3,
University of Almería, Carretera de Sacramento s/n, E-04120 Almería, Spain
Correspondence: [email protected] (V.V.); [email protected] (A.S.-C.);
Tel.: +34-958-637-206 (V.V. & A.S.-C.); Fax: +34-958-637-083 (V.V. & A.S.-C.)
Academic Editor: Manickam Sugumaran
Received: 30 March 2016; Accepted: 4 May 2016; Published: 11 May 2016
Abstract: Psidium guajava L. is widely used like food and in folk medicine all around the world.
Many studies have demonstrated that guava leaves have anti-hyperglycemic and anti-hyperlipidemic
activities, among others, and that these activities belong mainly to phenolic compounds, although it
is known that phenolic composition in guava tree varies throughout seasonal changes. Andalusia
is one of the regions in Europe where guava is grown, thus, the aim of this work was to study the
phenolic compounds present in Andalusian guava leaves at different oxidation states (low, medium,
and high). The phenolic compounds in guava leaves were determined by HPLC-DAD-ESI-QTOF-MS.
The results obtained by chromatographic analysis reported that guava leaves with low degree of
oxidation had a higher content of flavonols, gallic, and ellagic derivatives compared to the other
two guava leaf samples. Contrary, high oxidation state guava leaves reported the highest content
of cyanidin-glucoside that was 2.6 and 15 times higher than guava leaves with medium and low
oxidation state, respectively. The QTOF platform permitted the determination of several phenolic
compounds with anti-diabetic properties and provided new information about guava leaf phenolic
composition that could be useful for nutraceutical production.
Keywords: Psidium guajava L.; HPLC-DAD-ESI-QTOF-MS; phenolic compounds; gallic and ellagic
derivatives; flavonols; cyanidin-glucoside
1. Introduction
Psidium guajava (P. guajava) L., from the Myrtaceae family, is common throughout tropical and
subtropical areas [1] and Andalusia is one of the regions in Europe where guava is grown. Moreover,
it is widely used like food and in folk medicine all around the world. Many studies have demonstrated
that guava leaves have anti-hyperglycemic and anti-hyperlipidemic activities [2–4], among others,
and these biological activities have mainly been related to the phenolic compounds [5].
Nowadays, alternative therapeutic strategies based on the use of phenolic compounds in food
products as “functional foods” and “nutraceuticals” are being developed. In fact, the capacity of
plant-derived foods to reduce the risk of chronic diseases has been demonstrated [6].
Int. J. Mol. Sci. 2016, 17, 699; doi:10.3390/ijms17050699
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 699
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It is known that P. guajava L. shows different phenological stages throughout its vegetative period
in response to environmental conditions [7], because of that, it has been seen that the accumulation
of specific compounds such as anthocyanins changes [8]. Furthermore, the response of the different
classes of phenolic compounds, especially flavonoids, also vary substantially [9]. This fact plays an
important role in finding the best conditions of the leaf in order to obtain the best recovery of the
target compounds for the development of a promising alternative source for ameliorating diabetes
complications [2–4].
In this sense, spectrophotometric analyses are still helpful for a preliminary identification and
quantification [10]; however, LC-MS has opened up new approaches for the structural characterization
of target compounds. Moreover, LC-TOF-MS can provide tentative identification of unknown peaks,
due to accurate-mass measurement [11]. So, both UV-VIS diode array and mass spectrometry coupled
to HPLC have been proved as most appropriate analytical techniques for phenolic compounds in
many matrices [10,11]. Concerning guava leaves, most of the literature shows that quantification of
the different classes of phenolic compounds is generally done via spectrophotometric analysis [12–15],
although different analytical techniques, such as LC-DAD and LC-DAD-MS, are used to characterize
the bioactive compounds present in guava leaves [3,4,16,17].
Despite these facts and to our knowledge, there is no literature taking into account the change
that climatic conditions cause in phenolic composition and this information would be useful to choose
the best raw material for nutraceutical scopes. Thus, the aim of this work was to study the phenolic
compounds present in Andalusian guava leaves at different oxidation states (low, medium, and high)
by HPLC-DAD-ESI-QTOF-MS.
2. Results and Discussion
2.1. Characterization of Phenolic Compounds
The HPLC-DAD-ESI-QTOF-MS analyses in negative mode permitted the identification and
quantification of seventy-three phenolic compounds in guava leaves [18]. The individual compounds
were quantified on the basis of their peak area and compared with calibration curves obtained with the
corresponding standards and then expressed as µg/g of leaf dry weight (d.w.). Moreover, quantification
of compounds for which no commercial standards were available, was achieved comparing with
standard compounds bearing similar structures (Table 1).
Furthermore, the analysis in positive mode allowed the identification of an anthocyanin
compound. The compound, with m/z 449.1090 presented its maximum of absorption at 280, 350, and
520 nm on the UV spectrum. The MS/MS analysis produced a fragment ion at 287 m/z corresponding
to the loss of hexose unit (Figure 1). Due to the UV and MS spectrum and by co-elution with a
commercial standard, this component has been identified as cyanidin-3-O-glucoside. To our knowledge,
this compound has been identified for the first time in guava leaves. Limits of detection (LOD) and
quantification (LOQ) calculated for the cyanidin-3-O-β-galactopyranoside standard were 0.007 and
0.024 mg/L.
In terms of concentration of the individual compounds (Table 1), in negative mode, leaves with
lower oxidation state exhibited the highest amounts for almost all compounds quantified, followed by
moderate oxidized leaves and, finally, highly oxidized leaves. Concentrations of several compounds
tentatively identified [18], such as procyanidin tetramer and pentamer, galloyl-(epi)catechin trimer
isomers 1 and 2, and quercetin glucuronide were found to be lower than the quantification limit
for all samples. In contrast, and as it was expected due to the red coloration of the leaves at
high oxidative state, in positive mode, opposite results were found (Table 1); the concentration
of cyanidin-3-O-glucoside increased as the oxidation state of the leaves increased. At low oxidative
state, the concentration of cyanidin-3-O-glucoside was 29.5 ˘ 0.2 µg/g leaf d.w. and it raised until
441.28 ˘ 0.04 µg/g leaf d.w. for the highest oxidation state.
Int. J. Mol. Sci. 2016, 17, 699
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Table 1. Quantification (mean ˘ standard deviation (SD), n = 3) by HPLC-DAD-ESI-QTOF-MS in
negative and positive mode of individual compound tentatively identified in P. guajava leaves for the
different oxidative states.
No.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Negative mode
HHDP glucose Isomer
HHDP glucose Isomer
HHDP glucose Isomer
Prodelphinidin B Isomer
Gallic acid
Pedunculagin/Casuariin Isomer
Pedunculagin/Casuariin Isomer
Prodelphinidin Dimer Isomer
Gallocatechin
Vescalagin/castalagin Isomer
Prodelphinidin Dimer Isomer
Uralenneoside
Geraniin Isomer
Pedunculagin/Casuariin Isomer
Geraniin Isomer
Procyanidin B Isomer
Galloyl(epi)catechin-(epi)gallocatechin
Procyanidin B Isomer
Tellimagrandin I Isomer
Pterocarinin A Isomer
Pterocarinin A Isomer
Stenophyllanin A
Procyanidin trimer Isomer
Catechin
Procyanidin tetramer
Procyanidin trimer Isomer
Guavin A
Casuarinin/Casuarictin Isomer
Galloyl(epi)catechin-(epi)gallocatechin
Procyanidin pentamer
Galloyl-(epi)catechin trimer Isomer
Gallocatechin
Tellimagrandin I Isomer
Vescalagin
Stenophyllanin A Isomer
Galloyl-(epi)catechin trimer Isomer
Myricetin hexoside Isomer
Stachyuranin A
Procyanidin gallate Isomer
Myricetin hexoside Isomer
Vescalagin/castalagin Isomer
Myricetin arabinoside/xylopyranoside Isomer
Myricetin arabinoside/xylopyranoside Isomer
Procyanidin gallate Isomer
Myricetin arabinoside/xylopyranoside Isomer
Myricetin hexoside Isomer
Myricetin hexoside Isomer
Myricetin arabinoside/xylopyranoside Isomer
Quercetin galloylhexoside Isomer
Ellagic acid deoxyhexoside
High
Medium
Low
Concentration (µg compound/g leaf d.w.)
526 ˘ 2 c
936 ˘ 10 a
651 ˘ 19 b
c
b
505 ˘ 3
823 ˘ 16 a
645 ˘ 3
c
b
510 ˘ 11
934 ˘ 2 a
645 ˘ 20
c
b
447.1 ˘ 0.1
715 ˘ 13 a
515.7 ˘ 0.4
c
b
153.52 ˘ 0.09
175.9 ˘ 0.7 a
164 ˘ 3
b
b
175 ˘ 3 a
158.8 ˘ 0.6
163.84 ˘ 0.06
464.0 ˘ 0.8 c
557 ˘ 2 a
475.5 ˘ 0.5 b
603 ˘ 30 a
497 ˘ 1 b
529 ˘ 6 b
4913 ˘ 47 a
4098 ˘ 84 c
4435 ˘ 7 b
a
c
157.59 ˘ 0.01
136.6 ˘ 0.3
143 ˘ 2 b
c
b
1365 ˘ 7
1739 ˘ 25 a
1560 ˘ 14
a
b
2464 ˘ 4
1911 ˘ 24
1872 ˘ 81 b
b
b
343 ˘ 25 a
241 ˘ 1
264.9 ˘ 0.5
466 ˘ 3 c
683 ˘ 20 a
575 ˘ 16 b
356 ˘ 48 a
260 ˘ 3 b
290 ˘ 7 a,b
c
b
4262 ˘ 12
5514 ˘ 69 a
4742 ˘ 15
b
<LOQ
38 ˘ 3 a
12.60 ˘ 0.07
c
b
650 ˘ 3
757 ˘ 23 a
708 ˘ 11
c
b
347 ˘ 4
397 ˘ 2 a
367.2 ˘ 0.7
b
b
679 ˘ 7 a
569 ˘ 31
617 ˘ 9
b
316 ˘ 2 c
376 ˘ 4 a
360 ˘ 4
853 ˘ 13 c
1318 ˘ 24 a
1036 ˘ 50 b
781 ˘ 1 a
706 ˘ 1 c
738 ˘ 4 b
a
b
8957 ˘ 11
6845 ˘ 24 c
8486 ˘ 10
<LOQ
<LOQ
<LOQ
89 ˘ 2 c
128 ˘ 1 a
108 ˘ 3 b
263 ˘ 9 c
518 ˘ 15 a
357 ˘ 8 b
b
1297 ˘ 5 c
2089
˘ 11 a
1568 ˘ 10
61 ˘ 5 c
211 ˘ 12 a
135 ˘ 1 b
<LOQ
<LOQ
<LOQ
<LOQ
<LOQ
<LOQ
1526 ˘ 2 c
2613 ˘ 55 a
2074 ˘ 2 b
b
463 ˘ 2 c
737 ˘ 24 a
516 ˘ 6
187 ˘ 1 a
160 ˘ 6 b
159 ˘ 3 b
355.36 ˘ 0.07 c
548 ˘ 20 a
425 ˘ 13 b
<LOQ
<LOQ
<LOQ
432.10 ˘ 0.05 c
572 ˘ 7 a
555 ˘ 2 b
207.40 ˘ 0.04 a
207 ˘ 6 a
216 ˘ 1 a
533.2 ˘ 0.07 c
1036 ˘ 32 a
799 ˘ 3 b
213 ˘ 2 c
307.8 ˘ 0.8 a
288 ˘ 2 b
b
b
191 ˘ 3 a
152 ˘ 3
155 ˘ 2
c
b
241 ˘ 5
306 ˘ 5 a
286 ˘ 2
c
b
608 ˘ 1
946 ˘ 11 a
839 ˘ 8
a
b
11 ˘ 1
<LOQ
3.7 ˘ 0.2
688 ˘ 16 c
874 ˘ 9 a
816.0 ˘ 0.5 b
1186 ˘ 13 a
1010 ˘ 3 b
1012 ˘ 65 b
224 ˘ 6 a
200 ˘ 3 b
208 ˘ 5 b
282 ˘ 8 a
276.0 ˘ 0.9 a,b
266 ˘ 3 b
b
b
438 ˘ 18 a
375.0 ˘ 0.6
380 ˘ 5
a
a
700 ˘ 1
702 ˘ 12
733 ˘ 32 a
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Table 1. Cont.
No.
Compound
71
72
73
Negative mode
Quercetin galloylhexoside Isomer
Myricetin arabinoside/xylopyranoside Isomer
Morin
Myricetin arabinoside/xylopyranoside Isomer
Ellagic acid
Hyperin
Quercetin glucuronide
Isoquercitrin
Procyanidin gallate Isomer
Reynoutrin
Guajaverin
Guavinoside A
Avicularin
Quercitrin
Myrciaphenone B
Guavinoside C
Guavinoside B
Guavinoside A Isomer
Guavinoside B Isomer
2,6-dihydroxy-3-methyl-4-O-(6”-O-galloyl-β-Dglucopyranosyl)-benzophenone
Guavin B
Quercetin
Naringenin
74
Positive mode
Cyanidin-3-O-glucoside
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
High
Medium
Low
Concentration (µg compound/g leaf d.w.)
194 ˘ 7 a
205 ˘ 1 a
180 ˘ 2 b
b
b
588 ˘ 18 a
544.3 ˘ 0.4
525 ˘ 2
b
2619 ˘ 4 c
4474
˘ 98 a
3206 ˘ 11
611 ˘ 4 a
559 ˘ 3 c
581 ˘ 6 b
1229 ˘ 26 c
1759.6 ˘ 0.9 a
1345 ˘ 34 b
c
b
11305 ˘ 27
12528 ˘ 83 a
11906 ˘ 57
<LOQ
<LOQ
<LOQ
3410 ˘ 38 a
2254 ˘ 10 b
2471 ˘ 16 b
<LOQ
73.3 ˘ 0.2 a
7.3 ˘ 0.3 b
3210 ˘ 104 a
2641 ˘ 11 b
2762 ˘ 2 b
11813 ˘ 64 a
8864 ˘ 8 b
9668 ˘ 64 b
a,b
b
793 ˘ 4 a
783 ˘ 5
770 ˘ 4
a,b
b
11441 ˘ 63 a
10353 ˘ 18
10173 ˘ 54
b
b
223 ˘ 1 a
213 ˘ 2
208 ˘ 2
c
b
546 ˘ 6
715 ˘ 20 a
621 ˘ 1
c
b
1966 ˘ 21
2209 ˘ 21 a
2069 ˘ 1
b
872 ˘ 17 c
1273 ˘ 30 a
1035 ˘ 23
137 ˘ 1 a
135.1 ˘ 0.6 a
137 ˘ 3 a
b
b
129 ˘ 1 a
120 ˘ 2
119,6 ˘ 0.2
1179 ˘ 12 b
1242 ˘ 33 b
1365 ˘ 20 a
220.51 ˘ 0.03 b
258 ˘ 4 a
487 ˘ 3 c
230.1 ˘ 0.7 a,b
253 ˘ 3 a
638 ˘ 24 b
241 ˘ 7 a
255 ˘ 5 a
705 ˘ 6 a
Concentration (µg compound/g leaf d.w.)
441.28 ˘ 0.04 a
29.5 ˘ 0.2 c
169.3 ˘ 0.5 b
LOQ: limits of quantification; means in the same line with different letter (a,b,c ) are significantly different
(p < 0.05).
Int. J. Mol. Sci. 2016, 17, 699
Int. J. Mol. Sci. 2016, 17, 699
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Figure
Figure1.1.Fragmentation
Fragmentationpattern
patternofofcyanidin-3-O-glucoside.
cyanidin-3-O-glucoside.MS/MS
MS/MSspectra
spectrahas
hasbeen
beenobtained
obtainedby
byauto
autoMS/MS
MS/MS fragmentation.
Int. J. Mol. Sci. 2016, 17, 699
Int. J. Mol. Sci. 2016, 17, 699
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Several
Several works
works reported
reported the
the quantification
quantification of
of some
some compounds
compounds such
such as
as ellagic
ellagic acid,
acid, quercetin,
quercetin,
gallic
in in
guava
leaves
[3,9,12,19].
Comparing
the
gallic acid,
acid, catechin,
catechin,and
andgallocatechin
gallocatechinbybyHPLC-DAD
HPLC-DAD
guava
leaves
[3,9,12,19].
Comparing
results,
compounds
in
dried
leaves
are
found
in
lower
quantities
than
in
the
extract
[3,12],
the results, compounds in dried leaves are found in lower quantities than in the extract [3,12],
although
although values
values in
in the
the same
same order
order of
of magnitude
magnitude are
are noticed
noticed for
for quercetin
quercetin [9]
[9] and
and greater
greater amounts
amounts
of
of catechin
catechin and
and gallocatechin
gallocatechin are
are found
found comparing
comparing values
values with
with those
those obtained
obtained for
for dried
dried leaves
leaves
from
Korea
[19].
Furthermore,
Zhu
and
coworkers
[20]
also
isolated
the
major
compounds
from Korea [19]. Furthermore, Zhu and coworkers [20] also isolated the major compounds in
in
Chinese
guava
leaves:hyperoside,
hyperoside,isoquercitrin,
isoquercitrin, reynoutrin,
reynoutrin, guajaverin,
guajaverin, avicularin,
avicularin, and
Chinese
guava
leaves:
and also
also
2,4,6-trihydroxy-3,5-dimethylbenzophenone
4-O-(6″-O-galloyl)-βD
-glucopyranoside.
Concentrations
2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6”-O-galloyl)-β-D-glucopyranoside. Concentrations
of
of reynoutrin,
reynoutrin, guajaverin,
guajaverin, and
and avicularin
avicularin isomers
isomers followed
followed the
the same
same order
order as
as in
in the
the present
present
leaves
leaves (reynoutrin
(reynoutrin << guajaverin
guajaverin << avicularin)
avicularin) and
and the
the opposite
opposite order
order was
was observed
observed for
for the
the other
other
isomers
(isoquercitrin
<
hyperin).
The
compound
2,4,6-trihydroxy-3,5-dimethylbenzophenone
isomers (isoquercitrin < hyperin). The compound 2,4,6-trihydroxy-3,5-dimethylbenzophenone
4-O-(6″-O-galloyl)-β4-O-(6”-O-galloyl)-β-DD-glucopyranoside
-glucopyranosidewas
was not
not detected
detected inin the
the present
present samples,
samples, in
in contrast,
contrast,
2,6-dihydroxy-3-methyl-4-O-(6″-O-galloyl-βD-glucopyranosyl)-benzophenone
2,6-dihydroxy-3-methyl-4-O-(6”-O-galloyl-βD -glucopyranosyl)-benzophenone could
could be
be found.
found.
Differences
noticed
between
these
works
could
be
because
phenolic
composition
vary
substantially
Differences noticed between these works could be because phenolic composition vary substantially
among
among genotypes,
genotypes, seasons
seasons changes,
changes, ages,
ages, and
and damaged
damaged leaves,
leaves, and
and location
location sites
sites [7].
[7]. Regarding
Regarding the
the
different
families
present
in
leaves,
extracts
reported
significant
differences
(p
<
0.05),
the
lowest
different families present in leaves, extracts reported significant differences (p < 0.05), the lowest content
content
of the different
of phenolic
compounds
were found
in leaves
the highest
oxidation
of the different
classes classes
of phenolic
compounds
were found
in leaves
at the at
highest
oxidation
state,
state,
whereas
the
highest
content
was
found
in
leaves
at
the
lowest
oxidation
state
(Figure
2). class
The
whereas the highest content was found in leaves at the lowest oxidation state (Figure 2). The major
major
class compounds
of phenolic compounds
in guava
leaves
samples was
that ranged
between
48.1
of phenolic
in guava leaves
samples
was flavonols
that flavonols
ranged between
48.1 and
50.6 mg/g
and
50.6
mg/g
leaf
d.w.
The
second
class
of
polar
compounds
was
represented
by
flavan-3-ols
leaf d.w. The second class of polar compounds was represented by flavan-3-ols (24.2–24.7 mg/g
(24.2–24.7
leaf by
d.w.),
followed
by gallic
ellagic acid
derivatives
leaf d.w.)
leaf d.w.), mg/g
followed
gallic
and ellagic
acid and
derivatives
(14.8–15.8
mg/g(14.8–15.8
leaf d.w.) mg/g
and finally,
by
and
finally,
by
flavanones,
that
varied
from
0.49
to
0.63
mg/g
leaf
d.w.
Indeed,
the
contribution
of
flavanones, that varied from 0.49 to 0.63 mg/g leaf d.w. Indeed, the contribution of reynoutrin,
reynoutrin,
guajaverin,
and
avicularin
isomers
to
total
phenolic
content
(TPC)
was
predominant
in
guajaverin, and avicularin isomers to total phenolic content (TPC) was predominant in this work,
this
work, corresponding,
on25%
average,
to of
25%
of the
followed
by hyperinwhich
and
corresponding,
on average, to
and 26%
the and
TPC,26%
followed
byTPC,
hyperin
and isoquercitrin,
isoquercitrin,
which
supposed
about
15%.
Furthermore,
myricetin
derivatives
contributed
on
5%
to
supposed about 15%. Furthermore, myricetin derivatives contributed on 5% to TPC, morin and
TPC,
morin
and
quercetin
account
between
3%
and
5%
of
TPC
from
the
different
samples.
quercetin account between 3% and 5% of TPC from the different samples. Moreover, catechin was
Moreover,
catechin
between 7%isomers
and 10%,
gallocatechin
varied
between
7% and
ranged between
7%was
andranged
10%, gallocatechin
varied
between isomers
7% and 8%
of the
total amount,
8%
of
the
total
amount,
and
procyanidin
B
isomers
represented
a
6%.
and procyanidin B isomers represented a 6%.
Concentration (mg/g leaf d.w.)
60
gallic and ellagic derivatives
flavanones
b
b
50
flavonols
flavan-3-ols
a
40
30
20
a
b
c
a
a
b
10
c
b
a
0
high
medium
low
Figure 2.
families
of phenolic
compounds
present
in guava
different
Figure
2. Quantification
Quantificationofofdifferent
different
families
of phenolic
compounds
present
in leaves
guavaatleaves
at
oxidative
states.
The
different
letter
(a,b,c)
in
the
same
phenolic
class
means
a
significant
difference
different oxidative states. The different letter (a,b,c) in the same phenolic class means a significant
(p ď 0.05). (p ≤ 0.05).
difference
The variance
of the
individual
compounds
(Table(Table
1), and1),
theand
differences
between
The
varianceininconcentration
concentration
of the
individual
compounds
the differences
the families
2) (Figure
is probably
from
the different
synthesis
of secondary
as
between
the(Figure
families
2) isresulting
probably
resulting
from the
different
synthesis metabolites
of secondary
metabolites as response to the oxidative state of the leaves [9] and it could be explained by the
Int. J. Mol. Sci. 2016, 17, 699
7 of 13
response to the oxidative state of the leaves [9] and it could be explained by the formation of the
compounds in the flavonoid pathway [21,22]. Most of the compounds present in guava leaves derivate
from dihydroquercetin, precursor of both anthocyanins and flavonols. In fact, it has been noticed
that leaves with low oxidation state presented greater amounts of flavonols, such as quercetin and
myricetin derivatives, whereas the contents of the flavan-3ols catechin and gallocatechin, and the
content of cyanidin-3-O-glucoside were lower. In contrast, the highest oxidative state showed lower
amounts of quercetin and myricetin derivatives than low oxidation state, and higher concentration of
catechin, gallocatechin, and cyanidin-3-O-glucoside than low oxidized leaves (Table 1). Chang et al. [16]
also found for guava budding leaf tea that the presence of quercetin and its glycosides was larger than
catechins and myricetin derivatives. Moreover, the increasing concentration of cyanidin-3-O-glucoside
explained the dramatic red coloration of the leaves at higher oxidative states [21–23].
P. guajava leaves have traditionally been used in many countries to manage, control, and treat
the diabetes [24], and its potential against diabetes mellitus type 2 has also been demonstrated in
several works by the different parts of the plant, such as fruit, peel, pulp, seeds, and stem bark [25–28].
Singh et al. [29] reported that flavonoids are one of the major chemical constituents of plant species
used in the management of diabetic complications. In fact, the anti-diabetic activity has mainly been
attributed to a synergistic effect of the phenolic compounds present in the leaves [30]. This effect
has been observed in half ripen guava fruit for in vitro and in vivo assays [25]. Authors reported
that the effect is due to the total phenolic and flavonoid content in the fruit that was 40.13 ˘ 2.12
and 18.43 ˘ 1.22 mg/g of dry weight sample, respectively. Moreover, the recovery of total phenolic
compounds in the peel was 58.7 ˘ 4.0 g gallic acid equivalent (GAE)/kg dry matter [31]. Ribeiro
da Silva and coworkers [32] reported that the phenolic contents in the pulp was 1723.06 ˘ 111.58 mg
GAE/100 g dry basis; in the seeds varied between 14.54 and 91.05 mg total phenols (TP)/100 g
of defatted ground seeds in several solvents [33]; and the TPC of the stem bark, determined by
Folin–Ciocalteu, was 1.15 ˘ 0.12 g GAE/100 g dry weight [34]. These values are lower than the
ones summarized in the present work, so it could be supposed that guava leaves could be better
anti-diabetic agents than the other parts of the plant.
Comparing the phenolic content of guava leaves to other plant leaves, different evidence has
been found. The ethanol extract from Telfairia occidentalis leaves has also exhibited anti-hyperglycemic
activity and has demonstrated strong inhibition of α-glucosidase and mild inhibition of α-amylase [35].
Authors related its activity to the phenolic and flavonoid content; however, its content was less than
three order of magnitude than that reported in the present work. Additionally, other plants such as
Teucrium polium, cinnamon and garlic, have generally presented lower phenolic contents than guava
leaves [36–38] are also widely used in folk medicine for the treatment of diabetes [25]. In fact, different
extracts from Teucrium polium (leaves, flowers, and stems) reported a TPC between 14.6 to 157.8 mg of
GAE/g of extract [36], different parts of Cinnamomum cassia (barks, buds, and leaves) exhibited values
for TPC from 6.3 to 9.5 g/100 g d.w. [37], and 3.4–10.8 mg GAE/g d.w. was the range of TPC found for
different cultivars of garlic [38]. Additionally, several individual compounds isolated from different
sources that have also been found in guava leaves have demonstrated anti-diabetic properties (Table 2).
The principal activities related to these compounds are the inhibition of carbohydrate-hydrolysing
enzymes due to the presence of myrciaphenone B [39], casuarictin and tellimagrandin I [40],
flavonol glycosides (hyperin, isoquercitrin, reynoutrin, guajaverin, avicularin) [30,41], geraniin and
catechin [42], quercetin [30], and cyanidin-3-O-β-glucoside [43]. Insulinomimetic activity has been
attached to casuarinin, casuariin [40], procyanidin oligomers [44], and pedunculagin [45]. Moreover,
geraniin, vescalagin, gallic acid, naringenin, morin, quercetin, catechin, epicatechin, and procyanidin
B2 [42] exhibited anti-glycation activity, due to the inhibition of the formation of Amadori products and
advanced glycation end-products (AGEs). At last, the improvement of postprandial hyperglycemia
has been related to catechin and gallocatechin, among others [46].
Int. J. Mol. Sci. 2016, 17, 699
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Table 2. Guava leaves’ bioactive compounds related with anti-diabetic properties.
Compound
Assay
Myrciaphenone B
in vivo
Inhibition of aldose reductase α-glucosidase
Casuarictin, tellimagrandin I
in vitro
Inhibition of α-glucosidase
[40]
Cyanidin-3-O-β-glucoside
in vitro/in silico
Inhibition of α-amylase
[43]
Flavonol glycosides
in vitro
Inhibition of dipeptidyl-peptidase IV, and α-glucosidase
and α-amylase
Geraniin
in vitro
Hypoglycemic activity; inhibition of carbohydrate-hydrolysing
enzymes (α-glucosidase and α-amylase); effective in preventing
advanced glycation end-products (AGEs) formation
[42]
Vescalagin
in vivo
Retard AGEs formation
[42]
Gallic acid
in vitro
Inhibitory effect on the formation of α-dicarbonyl compounds
and protein glycation: inhibitory effects on the production of
Amadori products and AGEs
Naringenin
in vitro
Anti-glycation activity
[42]
Morin
in vitro
Protective activity against glycation
[42]
Quercetin
in vitro
Inhibitory effect on protein glycation, on the formation of
α-dicarbonyl compounds, and on the production of Amadori
products and AGEs
[3,30,42]
Catechin
in vitro/in vivo/
clinical trial
Inhibitory effect on the formation of α-dicarbonyl compounds
and protein glycation: inhibitory effects on the production of
Amadori products and AGEs; improvement of
postprandial hyperglycaemia
[42,46]
Procyanidin B2
in vitro/in vivo
Casuarinin, casuariin
in vitro
Procyanidin oligomers
in vitro/in vivo
Pedunculagin
in vivo
Gallocatechin
clinical trial
Activity
Ref.
[39]
[30,41]
[3,42]
Inhibitory effects on the formation of AGEs
[42]
Inhibition of insulin-like glucose uptake
[40]
Insulinomimetic properties
[44]
Improvement sensitivity of insulin
[45]
Improvement of postprandial hyperglycaemia
[46]
2.2. Antioxidant Capacity and Total Phenolic Content
The evaluation of the antioxidant capacity is usually done comparing different methods in
order to take into account the large number of factors that can influence the antioxidant action [47].
The choice of the two methods used in this work were assessed based on their different mechanisms:
Trolox Equivalent Antioxidant Capacity (TEAC) assay estimates the ability to scavenge 2,21 -azinobis
(3-ethylbenzothiazoline-6-sulfonate) (ABTS‚+ ) radicals and ferric reducing capacity is evaluated by the
Ferric Reducing Antioxidant Power (FRAP) method. As is shown in Table 3, significant differences
have been found among the different oxidation states (p < 0.05), the lowest oxidation states reported the
highest values for TEAC and FRAP (3.1 ˘ 0.1 mM eq Trolox/mg leaf d.w. and 5.4 ˘ 0.1 mM FeSO4 /mg
leaf d.w., respectively) and decrease as the oxidation state increases. Comparing the results with those
reported by Tachakittirungrod and coworkers [48] for guava leaves, similar values were accomplished
for TEAC and higher values for FRAP. This can be due to the fact that phenolic compounds in Spanish
leaves are more involved in the mechanism of reduction of oxidized intermediates in the chain reaction.
Table 3. Comparison (mean ˘ SD, n = 3) among total phenolic content (TPC) by HPLC-DAD-ESIQTOF-MS, Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric Reducing Antioxidant Power
(FRAP) of Psidium guajava L. leaves at different oxidative states.
Oxidation State
TPC (mg/g leaf d.w.)
TEAC
(mM eq Trolox/mg leaf d.w.)
FRAP
(mM FeSO4 /mg leaf d.w.)
High
Medium
Low
87.91 ˘ 0.05 c
92.0 ˘ 0.4 b
103 ˘ 2 a
2.2 ˘ 0.2 c
2.44 ˘ 0.05 b
3.1 ˘ 0.1 a
3.69 ˘ 0.03 c
4.20 ˘ 0.06 b
5.4 ˘ 0.1 a
Means in the same column with different letter (a,b,c ) are significantly different (p < 0.05).
Int. J. Mol. Sci. 2016, 17, 699
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Moreover, high correlation was found between the antioxidant assays (r = 0.9978 and p < 0.001),
in concordance with the data obtained for guava leaves [49], fruits [50], and in 30 plant extracts
of industrial interest [51]. However, TEAC value seems to be higher than FRAP value for guava
leaves [12,14,48].
Quantification of total phenolic compounds by HPLC-DAD-ESI-QTOF-MS revealed that the three
extracts showed significant differences (p < 0.05) as is displayed in Table 3. The lowest oxidation state
provided the highest content of total phenolic compounds (103 ˘ 2 mg/g leaf d.w.), followed by the
medium and the highest oxidation state (92.0 ˘ 0.4 and 87.91 ˘ 0.04 mg/g leaf d.w., respectively).
The values obtained were lower than those reported by several authors that employed Folin–Ciocalteu
method to quantify total phenolic compounds [12,14]. Even though the variance noticed is not great,
it could be because the determination by HPLC presented only 50–60 percentage of total phenolic
content [12]. Additionally, high correlation among TPC by HPLC, FRAP, and TEAC assays were found
in the present work. In fact, positive correlation with r = 0.9921 (p < 0.001) was noticed between TPC
by HPLC and FRAP and r = 0.9867 (p < 0.001) for TPC by HPLC and TEAC. Good correlations were
also found between TPC by Folin–Ciocalteu and antioxidant activity was also noticed for guava leaves
in literature [12,14,48].
3. Materials and Methods
3.1. Plant Material and Sample Preparation
Fresh guava leaves were harvested in Motril, Spain (36˝ 441 43”N 3˝ 311 14”W). The leaves were
collected in February 2014 at different oxidation states (low, medium, and high) based on the
difference of leaf color according to Hao [8]. The samples were air-dried at room temperature,
ground, and extracted with ethanol:water 80/20 (v/v) by ultrasonics as was previously reported by
Díaz-de-Cerio et al. [18].
3.2. Antioxidant Capacity Analysis
Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric Reducing Antioxidant Power (FRAP)
analysis were used to measure the antioxidant capacity.
For TEAC, ABTS radical cation was generated by reacting ABTS stock solution with 2.45 mM
potassium persulfate in the dark at room temperature for 12–24 h before use. A calibration curve was
prepared with different concentrations of Trolox (0–20 µM). The absorbance of ABTS radical cation
was adjusted to 0.70 (˘0.02) at 734 nm, and its change was measured [52].
To evaluate the reducing power, FRAP reagent (containing 2,4,6-tripyridyl-S-triazine (TPTZ),
FeCl3 and acetate buffer) was prepared. An aqueous solution of Fe (II) was used for calibration
(12.5–200 µM). The reduction was measured at 593 nm [53].
Results are expressed as mM eq Trolox/mg leaf d.w. and mM FeSO4 /mg leaf d.w., respectively.
3.3. HPLC-DAD-ESI-QTOF-MS Analysis
Chromatographic analyses were performed using an HPLC Agilent 1260 series (Agilent
Technologies, Santa Clara, CA, USA) equipped with a binary pump, an online degasser, an autosampler,
a thermostatically-controlled column compartment, and a UV-VIS diode array detector (DAD).
The column was maintained at 25 ˝ C. Phenolic compounds from P. guajava L. leaves were separated
using a method previously reported by Gómez-Caravaca et al. [54], in positive mode, slightly modified.
Briefly, a fused-core Poroshell 120, SB-C18 (3.0 mm ˆ 100 mm, 2.7 µm) from Agilent Technologies
(Agilent Technologies, Palo Alto, CA, USA) was used. The mobile phases were water plus 1% acetic
acid (A) and acetonitrile (B). A multi-step linear gradient was applied as follows; 0 min, 5% B; 2 min,
7% B; 4 min, 9% B; 6 min, 12% B; 8 min, 15% B; 9 min, 16% B; 10 min, 17% B; 11 min, 17.5% B; 12 min,
18% B; 13 min, 100% B; 17 min, 100% B; 18 min, 5% B. The initial conditions were maintained for 5 min.
The sample volume injected was 3 µL and the flow rate used was 0.8 mL¨ min´1 .
Int. J. Mol. Sci. 2016, 17, 699
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MS analyses were carried out using a 6540 Agilent Ultra-High-Definition Accurate-Mass
Q-TOF-MS coupled to the HPLC, equipped with an Agilent Dual Jet Stream electrospray ionization
(Dual AJS ESI) interface, at the following conditions: drying gas flow (N2 ), 12.0 L/min; nebulizer
pressure, 50 psi; gas drying temperature, 370 ˝ C; capillary voltage, 3500 V; fragmentor voltage and
scan range were 3500 V and m/z 50–1700. In positive mode, auto MS/MS experiments were carried
out using the followings collision energy values: m/z 100, 40 eV; m/z 500, 45 eV; m/z 1000, 50 eV;
and m/z 1500, 55 eV.
Additionally, phenolic compounds were also analyzed in negative mode using the
chromatographic and the detection method described by Díaz-de-Cerio et al. [18].
Standard calibration curves for cyanidin-3-O-β-galactopyranoside, gallic acid, catechin, ellagic
acid, naringenin, and rutin were prepared in the range of concentrations from the limit of quantification
(LOQ) to 50 mg/L and five calibration points for each standard were run in triplicate (n = 3).
Integration and data elaboration were performed using MassHunter Workstation software
(Agilent Technologies).
3.4. Statistical Analysis
The results reported in this study are the averages of three repetitions (n = 3). Fisher’s least
significance difference (LSD) test and Pearson’s linear correlations, both at p < 0.05, were evaluated
using Statistica 6.0 (2001, StatSoft, Tulsa, OK, USA).
4. Conclusions
HPLC coupled to QTOF-MS detector, which provides a molecular formula and the MS/MS data,
permitted the analysis of the major phenolic compounds of guava leaves. The method performed in
negative mode has proven to be successful in determining 73 compounds in the different guava leaves.
Moreover, in positive mode, the analysis with QTOF analyzer and the co-elution with a standard
solution allowed the identification of the cyanidin-glucoside. To our knowledge the cyanidin-glucoside,
was identified for the first time in guava leaves. Quantification data, in negative mode, reported that
leaves with low oxidation state presented the highest concentration of these compounds and decreased
when the oxidation state raise. On the contrary, the state of oxidation affected significantly the cyanidin
content. In fact, highest amount was detected in the leaves with high oxidation state.
Guava leaves seem to be a good source of phenolic compounds with described anti-diabetic
properties since several compounds present in the leaves have been related for ameliorating the effects
of diabetes mellitus disease, although this content varies due to the oxidative state of the leaf, so further
studies should be carried out in order to evaluate the influence of the phenolic composition on the
bioactivity of the extract.
Acknowledgments: This work was funded by the project co-financed by FEDER-Andalucía 2007–2013
(Cod. 461100) and Diputación de Granada. The authors Ana María Gómez-Caravaca and Vito Verardo thank the
Spanish Ministry of Economy and Competitiveness (MINECO) for “Juan de la Cierva” post-doctoral contracts.
Author Contributions: Elixabet Díaz-de-Cerio carried out the experimental analyses, data interpretation and
manuscript writing; Ana María Gómez-Caravaca and Vito Verardo design the experimental plan and were
involved in the data interpretation and manuscript redaction; Alberto Fernández-Gutiérrez and Antonio
Segura-Carretero were the responsibly of the project and founded the financial sources, moreover, they helped in
the data interpretation.
Conflicts of Interest: The authors declare no conflict of interest.
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